Detailed transcriptional maps and translational maps of selected portions of the vaccinia virus genome were constructed. More than 16 early transcripts were characterized and six early genes were sequenced. These studies revealed that early mRNAs are colinear with the genome, unspliced, and have multiple closely spaced 5 foot ends. The nucleotide sequences for about 60 base pairs upstream of transcriptional initiation sites are extremely A+T rich and contain long runs of As and Ts. The eukaryotic A2TA3 poly(A) signal sequence was not found near the ends of the genes. Marker rescue and in vitro translation techniques were developed to map functional vaccinia virus genes. In this manner, thymidine kinase was mapped within HindIII J fragment and sequenced. The nature of 3 independent TK- mutants was revealed by nucleotide sequencing. Each had a nucleotide reiteration leading to a +1 frameshift. In vitro and transient assay systems were developed to define regulatory nucleotide sequences and isolate regulatory proteins.